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Abstract Background We applied comparative phylogenomics (whole genome comparisons of microbes using DNA microarrays combined with Bayesian-based phylogenies) to investigate S. pneumoniae isolates from West Africa, with the aim of providing insights into the pathogenicity and other features related to the biology of the organism. The strains investigated comprised a well defined collection of 58 invasive and carriage isolates that were sequenced typed and included eight different S. pneumoniae serotypes (1, 3, 5, 6A, 11, 14, 19 F and 23 F) of varying invasive disease potential. Results The core genome of the isolates was estimated to be 38% and was mainly represented by gene functional categories associated with housekeeping functions. Comparison of the gene content of invasive and carriage isolates identified at least eleven potential genes that may be important in virulence including surface proteins, transport proteins, transcription factors and hypothetical proteins. Thirteen accessory regions (ARs) were also identified and did not show any loci association with the eleven virulence genes. Intraclonal diversity (isolates of the same serotype and MLST but expressing different patterns of ARs) was observed among some clones including ST 1233 (serotype 5), ST 3404 (serotype 5) and ST 3321 (serotype 14). A constructed phylogenetic tree of the isolates showed a high level of heterogeneity consistent with the frequent S. pneumoniae recombination. Despite this, a homogeneous clustering of all the serotype 1 strains was observed. Conclusions Comparative phylogenomics of invasive and carriage S. pneumoniae isolates identified a number of putative virulence determinants that may be important in the progression of S. pneumoniae from the carriage phase to invasive disease. Virulence determinants that contribute to S. pneumoniae pathogenicity are likely to be distributed randomly throughout its genome rather than being clustered in dedicated loci or islands. Compared to other S. pneumoniae serotypes, serotype 1 appears most genetically uniform.

Abstract Background Although Haemophilus influenzae type b (Hib), Staphylococcus aureus and Moraxella catarrhalis are important causes of invasive and mucosal bacterial disease among children, co-carriage with Streptococcus pneumoniae during infancy has not been determined in West Africa. Methods Species specific PCR was applied to detect each microbe using purified genomic DNA from 498 nasopharyngeal (NP) swabs collected from 30 Gambian neonates every two weeks from 0 to 6 months and bi-monthly up to 12 months. Results All infants carried S. pneumoniae, H. influenzae and M. catarrhalis at several time points during infancy. S.pneumoniae co-colonized the infant nasopharynx with at least one other pathogen nine out of ten times. There was early colonization of the newborns and neonates, the average times to first detection were 5, 7, 3 and 14 weeks for S. pneumoniae, H. influenzae, M. catarrhalis and S. aureus respectively. The prevalence of S. pneumoniae, H. influenzae and M. catarrhalis increased among the neonates and exceeded 80% by 13, 15 and 23 weeks respectively. In contrast, the prevalence of S. aureus decreased from 50% among the newborns to 20% amongst nine-week old neonates. S. pneumoniae appeared to have a strong positive association with H. influenzae (OR 5.03; 95% CI 3.02, 8.39; p < 0.01) and M. catarrhalis (OR 2.20; 95% CI 1.29; p < 0.01) but it was negatively associated with S. aureus (OR 0.53; 95% CI 0.30, 0.94; p = 0.03). Conclusion This study shows early acquisition and high co-carriage of three important respiratory pathogens with S. pneumoniae in the nasopharyngeal mucosa among Gambian neonates and infants. This has important potential implications for the aetiology of respiratory polymicrobial infections, biofilm formation and vaccine strategies.

Abstract Background Reports of the etiology of bacteremia in children from Nigeria are sparse and have been confounded by wide spread non-prescription antibiotic use and suboptimal laboratory culture techniques. We aimed to determine causative agents and underlying predisposing conditions of bacteremia in Nigerian children using data arising during the introduction of an automated blood culture system accessed by 7 hospitals and clinics in the Abuja area. Methods Between September 2008 and November 2009, we enrolled children with clinically suspected bacteremia at rural and urban clinical facilities in Abuja or within the Federal Capital Territory of Nigeria. Blood was cultured using an automated system with antibiotic removing device. We documented clinical features in all children and tested for prior antibiotic use in a random sample of sera from children from each site. Results 969 children aged 2 months-5 years were evaluated. Mean age was 21 ± 15.2 months. All children were not systematically screened but there were 59 (6%) children with established diagnosis of sickle cell disease and 42 (4.3%) with HIV infection. Overall, 212 (20.7%) had a positive blood culture but in only 105 (10.8%) were these considered to be clinically significant. Three agents, Staphylococcus aureus (20.9%), Salmonella typhi (20.9%) and Acinetobacter (12.3%) accounted for over half of the positive cultures. Streptococcus pneumoniae and non-typhi Salmonellae each accounted for 7.6%. Although not the leading cause of bacteremia, Streptococcus pneumoniae was the single leading cause of all deaths that occurred during hospitalization and after hospital discharge. Conclusion S. typhi is a significant cause of vaccine-preventable morbidity while S. pneumoniae may be a leading cause of mortality in this setting. This observation contrasts with reports from most other African countries where non-typhi Salmonellae are predominant in young children. Expanded surveillance is required to confirm the preliminary observations from this pilot study to inform implementation of appropriate public health control measures.

Abstract Background Helicobacter pylori is an important agent of gastroduodenal disease in Africa and throughout the world. We sought to determine an optimum method for genotyping H. pylori strains from children and adults in The Gambia, West Africa. Results Virulence genes were amplified in 127 of 190 cases tested (121 adults and 6 children); each of 60 bacterial cultures, and 116 from DNA extracted directly from biopsies. The proportion of biopsies that were cagA+, the ratio of vacAs1/s2, and vacAm1/m2, and the proportion of mixed strain populations in individual subjects changed with age. Strains lacking virulence cagA and vacA genes and with apparently homogeneous (one predominant strain) infections were more common among infants than adults. Conclusions In order to detect the range of bacterial genotypes harbored by individual patients, direct PCR proved slightly superior to isolation of H. pylori by biopsy culture, but the techniques were complementary, and the combination of both culture and direct PCR produced the most complete picture. The seemingly higher virulence of strains from adult than infant infections in The Gambia merits further analysis.

Abstract Background Currently available tools cannot be used to distinguish between sub-species of the M. tuberculosis complex causing latent tuberculosis (TB) infection. M. africanum causes up to half of TB in West- Africa and its relatively lower progression to disease suggests the presence of a large reservoir of latent infection relative to M. tuberculosis. Methods We assessed the immunogenicity of the TbD1 region, present in M. africanum and absent from "modern" M. tuberculosis, in an ELISPOT assay using cells from confirmed M. africanum or M. tuberculosis infected TB patients without HIV infection in the Gambia. Results Antigens from the TbD1 region induced IFNγ responses in only 35% patients and did not discriminate between patients infected with M. africanum vs. M. tuberculosis, while PPD induced universally high responses. Conclusions Further studies will need to assess other antigens unique to M. africanum that may induce discriminatory immune responses.

Abstract We report a case of an infant who experienced exogenous re-infection of Streptococcus pneumoniae serotype 14 as a cause of recurrent meningitis after apparently successful antibiotic treatment with ceftriaxone. eBURST analysis revealed that isolates from the two episodes of meningitis belonged to hypervirulent ST63 and ST3321 clonal complexes respectively.

Abstract Background Streptococcus pneumoniae serotype 1 causes > 20% of invasive disease, among all age groups combined, in The Gambia. In contrast, it is rarely detected in carriage studies. This study compares the molecular epidemiology of S. pneumoniae serotype 1 causing invasive disease in The Gambia between 1996 and 2005 to those carried in the nasopharynx between 2004 and 2006. Results A total of 127 invasive and 36 nasopharyngeal carriage serotype 1 isolates were recovered from individuals of all age groups and were analyzed by serotyping, antibiotic susceptibility testing and MLST. MLST analysis revealed 23 different sequence types (STs), 18 of which were novel. The most prevalent clone among the 163 isolates was ST618 (70.5%), followed by ST3575 (7.4%), ST2084 (2.5%) and ST612 (2.5%). A single ST (ST618), previously shown to belong to the ST217 hypervirulent clonal complex, was frequent among carriage (61.1%) and invasive (72.7%) serotype 1 isolates. ST618 causing both paediatric and adult disease peaked annually in the hot dry season and caused outbreak in 1997 and 2002. Conclusion For over a decade, isolates of ST618 have been the dominant lineage among serotype 1 carriage and disease isolates circulating in the Gambia. This lineage shows similar epidemiological features to those of the meningococcus in the African meningitis belt being able to cause outbreaks of disease

Abstract Background The study describes the molecular epidemiology of Streptococcus pneumoniae causing invasive disease in Gambian children Methods One hundred and thirty-two S. pneumoniae isolates were recovered from children aged 2–29 months during the course of a pneumococcal conjugate vaccine trial conducted in The Gambia of which 131 were characterized by serotyping, antibiotic susceptibility, BOX-PCR and MLST. Results Twenty-nine different serotypes were identified; serotypes 14, 19A, 12F, 5, 23F, and 1 were common and accounted for 58.3% of all serotypes overall. MLST analysis showed 72 sequence types (STs) of which 46 are novel. eBURST analysis using the stringent 6/7 identical loci definition, grouped the isolates into 17 clonal complexes and 32 singletons. The population structure of the 8 serotype 1 isolates obtained from 4 vaccinated and 2 unvaccinated children were the same (ST 618) except that one (ST3336) of the isolates from an unvaccinated child had a novel ST which is a single locus variant of ST 618. Conclusion We provide the first background data on the genetic structure of S. pneumoniae causing IPD prior to PC7V use in The Gambia. This data will be important for assessing the impact of PC7V in post-vaccine surveillance from The Gambia.

Abstract Background IFN-γ Release Assays (IGRAs) have been licensed for the diagnosis of latent Mycobacterium tuberculosis infection (LTBI). Their performance may depend on assay format and may vary across populations and settings. We compared the diagnostic performance of an in-house T -cell and commercial whole blood-based IGRAs for the diagnosis of LTBI and TB disease in The Gambia. Methods Newly diagnosed sputum smear positive cases and their household contacts were recruited. Cases and contacts were bled for IGRA and contacts had a Mantoux skin test. We assessed agreement and discordance between the tests and categorized a contact's level of M. tuberculosis exposure according to where s/he slept relative to a case: the same room, same house or a different house. We assessed the relationship between exposure and test results by multiple logistic regression. Results In 80 newly diagnosed TB cases, the sensitivity of ELISPOT was 78.7% and for QFT-GIT was 64.0% (p = 0.047). Of 194 household contacts 57.1% and 58.8% were positive for ELISPOT and QFT-GIT respectively. The overall agreement between both IGRAs for LTBI in contacts was 71.4% and there was no significant discordance (p = 0.29). There was significant discordance between the IGRAs and TST. Neither IGRA nor TST had evidence of false positive results because of Bacille Calmette Guérin (BCG) vaccination. However, agreement between QFT-GIT and TST as well as discordance between both IGRAs and TST were associated with BCG vaccination. Both IGRAs responded to the M. tuberculosis exposure gradient and were positively associated with increasing TST induration (p = 0.003 for ELISPOT and p = 0.001 for QFT-GIT). Conclusion The ELISPOT test is more sensitive than the QFT-GIT for diagnosing TB disease. The two tests perform similarly in the diagnosis of LTBI in TB contacts. Significant discordance between the two IGRAs and between each and the TST remain largely unexplained.

Abstract Background Few studies on bacteraemia in Africa have been published. We aimed to prospectively identify the causative organisms of bacteraemia in The Gambia and their relation to clinical diagnoses, outcome and antimicrobial susceptibility. Methods Between November 2003 and February 2005 we studied those admitted to the Medical Research Council hospital who were suspected of having bacteraemia. We documented clinical features, outcome, pathogens identified and their susceptibility patterns, and searched for factors associated with bacteraemia. Results 871 patients were admitted and had a blood culture taken. The median age was 2 years (range 2 months to 80 years) and 36 of 119 tested were HIV positive; 54.5% were male. 297 (34%) had a positive result and 93 (10.7% overall) were considered a genuine pathogen. Those with bacteraemia were more likely to die in hospital (OR 2.79; 1.17–6.65, p = 0.017) and to have a high white cell count (WCC; OR 1.81;95% CI 1.09–3.02; p = 0.022). Three organisms accounted for 73% of bacteraemias: Streptococcus pneumoniae (45.2%), Staphylococcus aureus (18.3%) and Escherichia coli (9.7%) while non-typhoidal salmonellae (NTS) accounted for 8.6%. Antimicrobial susceptibility of S. pneumoniae was very high to penicillin (97.5%); high resistance was found to co-trimoxazole. S. aureus was generally highly susceptible to cloxacillin, gentamicin and chloramphenicol. E. coli and NTS were all susceptible to ciprofloxacin and mostly susceptible to gentamicin. Thirteen (33%) S. pneumoniae isolates were of serotypes contained in a 7-valent pneumococcal conjugate vaccine and 20 (51.3%) were of the same serogroup. Conclusion In The Gambia, those with bacteraemia are more likely than those without to die in hospital and to have a raised peripheral blood WCC. S. pneumoniae is the most common organism isolated. Introduction of a pneumococcal conjugate vaccine can be expected to lead to a reduction in disease incidence.

Abstract Background Studies in Africa investigating health-seeking behaviour by interviewing tuberculosis patients have revealed patient knowledge issues and significant delays to diagnosis. We aimed to study health-seeking behaviour and experience of those with cough in The Gambia and to identify whether they had tuberculosis. Methods During a round of a population under 3-monthly demographic surveillance, we identified people >10 years old who had been coughing ≥ 3 weeks. A questionnaire was administered concerning demographic data, cough, knowledge, health seeking, and experience at health facilities. Case finding utilised sputum smear and chest X-ray. Results 122/29,871 coughing individuals were identified. Of 115 interviewed, 93 (81%) had sought treatment; 76 (81.7%) from the health system. Those that visited an alternative health provider first were significantly older than those who visited the health system first (p = 0.03). The median time to seek treatment was 2 weeks (range 0 – 106). 54 (58.1%) made their choice of provider because they believed it was right. Of those who left the health system to an alternative provider (n = 13): 7 believed it was the best place, 3 cited cost and 2 failure to improve. 3 cases were identified by sputum analysis, 11 more by X-ray; all had visited the health system first. Total 'excess' cough time was 1079 person weeks. Conclusion The majority of people with cough in this population seek appropriate help early. Improved case detection might be achieved through the use of chest X-ray in addition to sputum smear.

Abstract Background The tuberculosis (TB) epidemic in Africa is on the rise, even in low-HIV prevalence settings. Few studies have attempted to identify possible reasons for this. We aimed to identify risk factors for pulmonary tuberculosis in those attending a general outpatients clinic in The Gambia, a sub-Saharan African country with relatively low HIV prevalence in the community and in TB patients. Methods We conducted a case control study at the Medical Research Council Outpatients' clinic in The Gambia. Pulmonary TB cases were at least 15 years old, controls were age and sex matched clinic attendees. Participants were interviewed using a structured questionnaire. Results 100 sputum smear positive TB cases and 200 clinic controls were recruited. HIV prevalence was 6.1% in cases and 3.3% in controls. Multivariable assessment of host factors showed that risk of TB was increased among the Jola ethnic group and smokers, and decreased in those in a professional occupation. Assessment of environmental factors showed an increased risk with household crowding, history of household exposure to a known TB case, and absence of a ceiling in the house. In a combined multivariable host-environment model, the risk of TB increased with crowding, exposure to a known TB case, as well as amongst the Jola ethnic group. Conclusion In The Gambia, household crowding and past household exposure to a known TB case are the standout risk factors for TB disease. Further research is needed to identify why risk of TB seems to differ according to ethnicity.

Abstract Background New tools are required to improve tuberculosis (TB) diagnosis and treatment, including enhanced ability to compare new treatment strategies. The ELISPOT assay uses Mycobacterium tuberculosis-specific antigens to produce a precise quantitative readout of the immune response to pathogen. We hypothesized that TB patients in The Gambia would have reduced ELISPOT counts after successful treatment. Methods We recruited Gambian adults with sputum smear and culture positive tuberculosis for ELISPOT assay and HIV test, and followed them up one year later to repeat testing and document treatment outcome. We used ESAT-6, CFP-10 and Purified Protein Derivative (PPD) as stimulatory antigens. We confirmed the reliability of our assay in 23 volunteers through 2 tests one week apart, comparing within and between subject variation. Results We performed an ELISPOT test at diagnosis and 12 months later in 89 patients. At recruitment, 70/85 HIV-negative patients (82%) were ESAT-6 or CFP-10 (EC) ELISPOT positive, 77 (90%) were PPD ELISPOT positive. Eighty-two cases (96%) successfully completed treatment: 44 (55%; p < 0.001) were EC ELISPOT negative at 12 months, 17 (21%; p = 0.051) were PPD ELISPOT negative. Sixty (73%) cured cases had a CFP-10 ELISPOT count decrease, 64 (78%) had an ESAT-6 ELISPOT count decrease, 58 (70%) had a PPD ELISPOT count decrease. There was a mean decline of 25, 44 and 47 SFU/2 × 105 cells for CFP-10, ESAT-6 and PPD respectively (p < 0.001 for all). Three of 4 HIV positive patients were cured, all 3 underwent ELISPOT reversion; all 4 not cured subjects (3 HIV-negative, 1 HIV positive) were ESAT-6, CFP-10 and PPD ELISPOT positive at 12 months. Conclusion Successful tuberculosis treatment is accompanied by a significant reduction in the M. tuberculosis-specific antigen ELISPOT count. The ELISPOT has potential as a proxy measure of TB treatment outcome. Further investigation into the decay kinetics of T-cells with treatment is warranted.

Introduction and Rationale The introduction of routine immunisation with pneumococcal conjugate vaccines (PCVs) in developing countries is expected to significantly reduce childhood deaths [1]. The 7-valent pneumococcal conjugate vaccine (PCV-7), [...]

Introduction The prevention of pneumococcal disease is a major international public health priority, especially in children in developing countries [1]. On the basis of a trial conducted in California in [...], Background: Introduction of pneumococcal conjugate vaccines (PCVs) of limited valency is justified in Africa by the high burden of pneumococcal disease. Long-term beneficial effects of PCVs may be countered by serotype replacement. We aimed to determine the impact of PCV-7 vaccination on pneumococcal carriage in rural Gambia. Methods and Findings: A cluster-randomized (by village) trial of the impact of PCV-7 on pneumococcal nasopharyngeal carriage was conducted in 21 Gambian villages between December 2003 to June 2008 (5,441 inhabitants in 2006). Analysis was complemented with data obtained before vaccination. Because efficacy of PCV-9 in young Gambian children had been shown, it was considered unethical not to give PCV-7 to young children in all of the study villages. PCV-7 was given to children below 30 mo of age and to those born during the trial in all study villages. Villages were randomized (older children and adults) to receive one dose of PCV-7 (11 vaccinated villages) or meningococcal serogroup C conjugate vaccine (10 control villages). Cross-sectional surveys (CSSs) to collect nasopharyngeal swabs were conducted before vaccination (2,094 samples in the baseline CSS), and 4-6, 12, and 22 mo after vaccination (1,168, 1,210, and 446 samples in CSS-1, -2, and -3, respectively). A time trend analysis showed a marked fall in the prevalence of vaccine-type pneumococcal carriage in all age groups following vaccination (from 23.7% and 26.8% in the baseline CSS to 7.1% and 8.5% in CSS-1, in vaccinated and control villages, respectively). The prevalence of vaccine-type pneumococcal carriage was lower in vaccinated than in control villages among older children (5 y to Conclusions: Vaccination of Gambian children reduced vaccine-type pneumococcal carriage across all age groups, indicating a 'herd effect' in non-vaccinated older children and adults. No significant serotype replacement was detected.